Natural history of murine gamma-herpesvirus infection

Murine gamma-herpesvirus 68 (MHV-68) is a natural pathogen of small rodents and insectivores (mice, voles and shrews). The primary infection is characterized by virus replication in lung epithelial cells and the establishment of a latent infection in B lymphocytes. The virus is also observed to persist in lung epithelial cells, dendritic cells and macrophages. Splenomegaly is observed two weeks after infection, in which there is a CD4+ T-cell-mediated expansion of B and T cells in the spleen. At three weeks post-infection an infectious mononucleosis-like syndrome is observed involving a major expansion of Vbeta4+CD8+ T cells. Later in the course of persistent infection, ca. 10% of mice develop lymphoproliferative disease characterized as lymphomas of B-cell origin. The genome from MHV-68 strain g2.4 has been sequenced and contains ca. 73 genes, the majority of which are collinear and homologous to other gamma-herpesviruses. The genome includes cellular homologues for a complement-regulatory protein, Bcl-2, cyclin D and interleukin-8 receptor and a set of novel genes M1 to M4. The function of these genes in the context of latent infections, evasion of immune responses and virus-mediated pathologies is discussed. Both innate and adaptive immune responses play an active role in limiting virus infection. The absence of type I interferon (IFN) results in a lethal MHV-68 infection, emphasizing the central role of these cytokines at the initial stages of infection. In contrast, type II IFN is not essential for the recovery from infection in the lung, but a failure of type II IFN receptor signalling results in the atrophy of lymphoid tissue associated with virus persistence. Splenic atrophy appears to be the result of immunopathology, since in the absence of CD8+ T cells no pathology occurs. CD8+ T cells play a major role in recovery from the primary infection, and also in regulating latently infected cells expressing the M2 gene product. CD4+ T cells have a key role in surveillance against virus recurrences in the lung, in part mediated through 'help' in the genesis of neutralizing antibodies. In the absence of CD4+ T cells, virus-specific CD8+ T cells are able to control the primary infection in the respiratory tract, yet surprisingly the memory CD8+ T cells generated are unable to inhibit virus recurrences in the lung. This could be explained in part by the observations that this virus can downregulate major histocompatibility complex class I expression and also restrict inflammatory cell responses by producing a chemokine-binding protein (M3 gene product). MHV-68 provides an excellent model to explore methods for controlling gamma-herpesvirus infection through vaccination and chemotherapy. Vaccination with gp150 (a homologue of gp350 of Epstein-Barr virus) results in a reduction in splenomegaly and virus latency but does not block replication in the lung, nor the establishment of a latent infection. Even when lung virus infection is greatly reduced following the action of CD8+ T cells, induced via a prime-boost vaccination strategy, a latent infection is established. Potent antiviral compounds such as the nucleoside analogue 2'deoxy-5-ethyl-beta-4'-thiouridine, which disrupts virus replication in vivo, cannot inhibit the establishment of a latent infection. Clearly, devising strategies to interrupt the establishment of latent virus infections may well prove impossible with existing methods.     

Signaling lymphocyte activation molecule-associated protein is a negative regulator of the CD8 T cell response in mice

The primary manifestation of X-linked lymphoproliferative syndrome, caused by a dysfunctional adapter protein, signaling lymphocyte activation molecule-associated protein (SAP), is an excessive T cell response upon EBV infection. Using the SAP-/- mouse as a model system for the human disease, we compared the response of CD8+ T cells from wild-type (wt) and mutant mice to various stimuli. First, we observed that CD8+ T cells from SAP-/- mice proliferate more vigorously than those from wt mice upon CD3/CD28 cross-linking in vitro. Second, we analyzed the consequence of SAP deficiency on CTL effector function and homeostasis. For this purpose, SAP-/- and wt mice were infected with the murine gamma-herpesvirus 68 (MHV-68). At 2 wk postinfection, the level of viral-specific CTL was much higher in mutant than in wt mice, measured both ex vivo and in vivo. In addition, we established that throughout 45 days of MHV-68 infection the frequency of virus-specific CD8+ T cells producing IFN-gamma was significantly higher in SAP-/- mice. Consequently, the level of latent infection by MHV-68 was considerably lower in SAP-/- mice, which indicates that SAP-/- CTL control this infection more efficiently than wt CTL. Finally, we found that the Vbeta4-specific CD8+ T cell expansion triggered by MHV-68 infection is also enhanced and prolonged in SAP-/- mice. Taken together, our data indicate that SAP functions as a negative regulator of CD8+ T cell activation.     

Quantitative analysis of the acute and long-term CD4(+) T-cell response to a persistent gammaherpesvirus

The murine gammaherpesvirus 68 (MHV-68) replicates in respiratory epithelial cells, where it establishes a persistent, latent infection limited predominantly to B lymphocytes. The virus-specific CD4(+) T-cell response in C57BL/6 mice challenged intranasally with MHV-68 is detected first in the mediastinal lymph nodes and then in the cervical lymph nodes and the spleen. The numbers of MHV-68-specific CD4(+) T cells generated in congenic mice homozygous for disruption of the beta2-microglobulin gene tended to be higher, indicating that the absence of the CD8(+) set in this group resulted in a compensatory response. The peak frequency within the splenic CD4(+) T-cell population may reach 1:50 in the acute response; it then drops to 1:400 to 1:500 within 4 months and stays at that level in the very long term. Sorting for L-selectin (CD62L) expression established that all virus-specific CD4(+) T cells were initially CD62Llow, with >80% maintaining that phenotype for the next 14 months. The overall conclusion is that MHV-68-specific CD4(+) T cells remain activated (CD62Llow) and at a stable frequency in the face of persistent infection.     

Dissecting the host response to a gamma-herpesvirus

The murine gamma-herpesvirus 68 (MHV-68) provides a unique experimental model for dissecting immunity to large DNA viruses that persist in B lymphocytes. The analysis is greatly facilitated by the availability of genetically disrupted (-/-) mice that lack key host-response elements, and by the fact that MHV-68 is a lytic virus that can readily be manipulated for mutational analysis. The mutant virus strategy is being used, for example, to characterize the part played in vivo by an MHV-68-encoded chemokine-binding protein that may ultimately find an application in human therapeutics. Experiments with various -/- mice and monoclonal antibody depletion protocols have shown very clearly that type I interferons (IFNs) are essential for the early control of MHV-68 replication, while CD4+ T cells producing IFN-gamma function to limit the consequences of viral persistence. Virus-specific CD8+ effectors acting in the absence of the CD4+ subset seem initially to control the lytic phase in the lung following respiratory challenge, but are then unable to prevent the reactivation of replicative infection in epithelia and the eventual death of CD4+ T-cell-deficient mice. This could reflect the fact that the interaction between the CD8+ T cells and the virus-infected targets is partially compromised by the MHV-68 K3 protein, which inhibits antigen presentation by MHC class I glycoproteins. Immunization strategies focusing on the CD8+ T-cell response to epitopes expressed during the lytic phase of MHV-68 infection can limit virus replication, but are unable to prevent the establishment of latency. Other experiments with mutant viruses also suggest that there is a disconnection between lytic MHV-68 infection and latency. The massive nonspecific immunoglobulin response and the dramatic expansion of Vbeta4+ CD8+ T cells, which is apparently MHC independent, could represent some sort of 'smoke screen' used by MHV-68 to subvert immunity. Although MHV-68 is neither Epstein-Barr virus nor human herpesvirus-8, the results generated from this system suggest possibilities that may usefully be addressed with these human pathogens. Perhaps the main lesson learned to date is that all the components of immunity are likely to be important for the control of these complex viruses.     

Suppressive CD8+ T cells arise in the absence of CD4 help and compromise control of persistent virus

There is an urgent need to develop novel therapies for controlling chronic virus infections in immunocompromised patients. Disease associated with persistent γ-herpesvirus infection (EBV, human herpesvirus 8) is a significant problem in AIDS patients and transplant recipients, and clinical management of these conditions is difficult. Immune surveillance failure followed by γ-herpesvirus recrudescence can be modeled using murine γ-herpesvirus (MHV)-68 in mice lacking CD4(+) T cells. In contrast with other chronic infections, no obvious defect in the functional capacity of the viral-specific CD8(+) T cell response was detected. We show in this article that adoptive transfer of MHV-68-specific CD8(+) T cells was ineffective at reducing the viral burden. Together, these indicate the potential presence of T cell extrinsic suppressive factors. Indeed, CD4-depleted mice infected with MHV-68 express increased levels of IL-10, a cytokine capable of suppressing the function of both APCs and T cells. CD4-depleted mice developed a population of CD8(+) T cells capable of producing IL-10 that suppressed viral control. Although exhibiting cell surface markers indicative of activation, the IL-10-producing cells expressed increased levels of programmed death-1 but were not enriched in the MHV-68-specific compartment, nor were they uniformly CD44(hi). Therapeutic administration of an IL-10R blocking Ab enhanced control of the recrudescent virus. These data implicate IL-10 as a promising target for the restoration of immune surveillance against chronic γ-herpesvirus infection in immunosuppressed individuals.     

IL-15-independent proliferative renewal of memory CD8+ T cells in latent gammaherpesvirus infection

IL-15 is known to be critical in the homeostasis of Ag-specific memory CD8(+) T cells following acute viral infection. However, little is known about the homeostatic requirements of memory CD8(+) T cells during a latent viral infection. We have used the murine gammaherpesvirus-68 (MHV-68) model system to investigate whether IL-15 is necessary for the maintenance of memory CD8(+) T cells during a latent viral infection. IL-15 is not essential either for the initial control of MHV-68 infection or for the maintenance of MHV-68-specific memory CD8(+) T cells. Even at 140 days postinfection, the proportion of CD8(+) T cells recognizing the MHV-68 epitopes were the same as in control mice. The maintenance of these memory CD8(+) T cells was attributable to their ability to turn over in vivo, probably in response to the presence of low levels of Ag. IL-15(-/-) mice had a significantly higher turnover rate within the virus-specific memory CD8(+) T cell population, which was the result of increased levels of viral gene expression rather than an increase in viral load. These cells did not accumulate in the spleens of the IL-15(-/-) mice due to an increased sensitivity to apoptosis as a result of decreased Bcl-2 levels. Intriguingly, memory CD8(+) T cells from latently infected mice failed to undergo homeostatic proliferation in a naive secondary host. These data highlight fundamental differences between memory CD8(+) T cells engaged in active immune surveillance of latent viral infections vs memory CD8(+) T cells found after acute viral infections.     

Protein kinase C theta is not essential for T-cell-mediated clearance of murine gammaherpesvirus 68

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen with significant homology to human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. T cells are essential for primary clearance of MHV-68 and survival of mice following intranasal infection. Previous reports have suggested that protein kinase C theta (PKCtheta) is essential for T-cell activation and cytokine production in vitro. To determine the role of this molecule in vivo during the immune response to a viral infection, PKCtheta-/- mice were infected with MHV-68. Despite the essential role of T cells in viral clearance, PKCtheta-/- mice survived infection, cleared lytic virus, and maintained effective long-term control of latency. CD8 T-cell expansion, trafficking to the lung, and cytotoxic activity were similar in PKCtheta+/+ and PKCtheta-/- mice, whereas antiviral antibody and T-helper cell cytokine production were significantly lower in PKCtheta-/- mice than in PKCtheta+/+ mice. These studies demonstrate a differential requirement for PKCtheta in the immune response to MHV-68 and show that PKCtheta is not essential for the T-cell activation events leading to viral clearance.     

Control of memory CD8+ T cell differentiation by CD80/CD86-CD28 costimulation and restoration by IL-2 during the recall response

Memory CD8+ T cell responses have been considered to be independent of CD80/CD86-CD28 costimulation. However, recall responses are often severely blunted in CD28-/- mice. Whether this impairment represents a requirement for CD28 costimulation for proper memory CD8+ T cell development or a requirement during the recall response is unknown. Furthermore, how CD28 costimulation affects the phenotype and function of memory CD8+ T cells has not been characterized in detail. In this study, we investigate these questions by studying the role of the CD28 costimulatory pathway in memory CD8+ T cell responses to acute and persistent DNA virus infections. Memory CD8+ T cells against vaccinia virus (VV) infection which develop without CD28 costimulation exhibit lower expression of differentiation markers CD27 and CD122 (IL-15Rbeta). These memory CD8+ T cells also fail to produce IL-2. Our data indicate that for an optimal recall response, CD28 costimulation is required both for T cell priming and also during the recall response. Similar requirements were observed for memory CD8+ T cell responses during persistent infection with murine gammaherpesvirus 68 (MHV-68) infection, indicating CD28 may play the same role in both acute and persistent infections. Finally, we show deficits in the recall response are restored by IL-2 signaling during recall, but not during priming. The data presented show that CD28 costimulation not only controls the magnitude of the primary response but also affects development of memory CD8+ T cells and is required during the recall response in addition to initial T cell priming.     

Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gamma HV-68; also referred to as MHV-68) is a gammaherpesvirus which infects murid rodents. Previous studies showed that CD8 T cells are important for controlling gamma HV-68 replication during the first 2 weeks of infection and suggested a role for B cells in latent or persistent gamma HV-68 infection. To further define the importance of B cells and CD8 T cells during acute and chronic gamma HV-68 infection, we examined splenic infection in mice with null mutations in the transmembrane domain of the mu-heavy-chain constant region (MuMT; B-cell and antibody deficient) or in the beta2-microglobulin gene (beta2 -/-; CD8 deficient). Immunocompetent mice infected intraperitoneally with gamma HV-68 demonstrated peak splenic titers 9 to 10 days postinfection, cleared infectious virus 15 to 20 days postinfection, and harbored low levels of latent virus at 6 weeks postinfection. Beta2-/- mice showed peak splenic gamma HV-68 titers similar to those of normal mice but were unable to clear infectious virus completely from the spleen, demonstrating persistent infectious virus 6 weeks postinfection. These data indicate that CD8 T cells are important for clearing infectious gamma HV-68 from the spleen. Infected MuMT mice did not demonstrate detectable infectious gamma HV-68 in the spleen at any time after infection, indicating that mature B lymphocytes are necessary for acute splenic infection by gamma HV-68. Despite the lack of measurable acute infection, MuMT spleen cells harbored latent virus 6 weeks postinfection at a level about 100-fold higher than that in normal mice. These data demonstrate establishment of latency by a herpesvirus in an organ in the absence of acute viral replication in that organ. In addition, they demonstrate that gamma HV-68 can establish latency in a cell type other than mature B lymphocytes.     

A Primary Study of the Subgroups of T Lymphocytes in MHV-3 Induced Chronic Viral Hepatitis

To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type 3(MHV-3) induced chronic viral hepatitis in C3H/Hej mice,ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units(PFU)of MHV-3 intraperitoneally.The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin(HE) staining method from 2 days post MHV-3 infection.The ratios of T cell subsets including CD3 CD4 CD8-,CD3 CD4-CD8 ,CD3 CD4-CD8-,CD3 CD4 CD25 ,CD3 CD4 CD25-and CD3 CD4-CD25 T lymphocyte of total T lymphocytes in blood,spleen and liver were examined at 0,2,4,6,8,10,12,15,20,25,30,40 days post MHV-3 infection by flow cytosorting.We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection.The double negative T cell(DN Treg cell) and CD4 CD25 T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice,and CD3 CD4 CD8-,CD3 CD4-CD8 ,CD3 CD4 CD25-and CD3 CD4-CD25 T cell ratios decreased accordingly.In conclusion,the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence.Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice.The increase of the DN Treg cell and CD4 CD25 T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4 CD25 T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection.Further characterizations of DNT cell and CD4 CD25 T cell are under investigation.     

Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages

Intranasal infection of mice with the murine gamma-herpesvirus MHV-68 results in an acute lytic infection in the lung, followed by the establishment of lifelong latency. Development of an infectious mononucleosis-like syndrome correlates with the establishment of latency and is characterized by splenomegaly and the appearance of activated CD8+ T cells in the peripheral blood. Interestingly, a large population of activated CD8+ T cells in the peripheral blood expresses the V beta 4+ element in their TCR. In this report we show that MHV-68 latency in the spleen after intranasal infection is harbored in three APC types: B cells, macrophages, and dendritic cells. Surprisingly, since latency has not previously been described in dendritic cells, these cells harbored the highest frequency of latent virus. Among B cells, latency was preferentially associated with activated B cells expressing the phenotype of germinal center B cells, thus formally linking the previously reported association of latency gene expression and germinal centers to germinal center B cells. Germinal center formation, however, was not required for the establishment of latency. Significantly, although three cell types were latently infected, the ability to stimulate V beta 4+CD8+ T cell hybridomas was limited to latently infected, activated B cells.     

CD8+ T cell dysfunction and increase in murine gammaherpesvirus latent viral burden in the absence of 4-1BB ligand

Studies of costimulatory receptors belonging to the TNFR family have revealed their diverse roles in affecting different stages of the T cell response. The 4-1BB ligand (4-1BBL)/4-1BB pathway has emerged as a receptor-ligand pair that impacts not the initial priming, but later phases of the T cell response, such as sustaining clonal expansion and survival, maintaining memory CD8(+) T cells, and supporting secondary expansion upon Ag challenge. Although the role of this costimulatory pathway in CD8(+) T cell responses to acute viral infections has been well-studied, its role in controlling chronic viral infections in vivo is not known to date. Using the murine gammaherpesvirus-68 (MHV-68) model, we show that 4-1BBL-deficient mice lack control of MHV-68 during latency and show significantly increased latent viral loads. In contrast to acute influenza infection, the numbers of MHV-68-specific memory CD8(+) T cells were maintained during latency. However, the virus-specific CD8(+) T cells showed defects in function, including decreased cytolytic function and impaired secondary expansion. Thus, 4-1BBL deficiency significantly affects the function, but not the number, of virus-specific CD8(+) T cells during gammaherpesvirus latency, and its absence results in an increased viral burden. Our study suggests that the 4-1BB costimulatory pathway plays an important role in controlling chronic viral infections.     

Pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for CD8 T cells.

Murine gammaherpesvirus is a natural rodent pathogen which causes a primary infection in the lungs and establishes a persistent infection in B lymphocytes. During the primary infection, large amounts of gamma interferon (IFN-gamma) are produced by spleen, mediastinal, and cervical lymph node cells. To investigate the role of IFN-gamma in control of the virus infection, mice lacking the cellular receptor for IFN-gamma (IFN-gamma R-/- mice) were infected with murine gammaherpesvirus 68 (MHV68). IFN-gamma R-/- mice showed no difference from wild-type mice in the titers of infectious virus in the lungs or in the rate of clearance of the lung infection. In the spleen, however, clear differences were observed. By 14 days postinfection, spleens from IFN-gamma R-/- mice were pale, shrunken, and fibrous. Histological examination showed that there was an early (day 10) infiltration of granulocytes followed by widespread destruction of splenic architecture (days 14 to 17). A marked decrease in the number of splenic B cells and CD4+ and CD8+ T cells occurred. These changes were accompanied by a 10- to 100-fold greater load of latently infected cells in IFN-gamma R-/- mice than in wild-type mice at 14 to 17 days postinfection, but this was reduced to the levels found in wild-type mice by 21 days postinfection. Treatment of the mice with the antiviral drug 2'-deoxyl-5-ethyl-beta-4'-thiouridine from 6 days postinfection did not prevent the occurrence of these changes. The changes were, however, completely reversed by depletion of CD8+ T cells prior to and during the primary infection. Depletion of CD4+ T cells also reversed the major pathological and virological changes, although in this case there was evidence of some histological changes. Thus, the lack of IFN-gamma receptor had profound consequences in spleens of MHV68-infected mice. The possible mechanisms involved in these changes are discussed.     

CD8(+) T cells from mice transnuclear for a TCR that recognizes a single H-2K(b)-restricted MHV68 epitope derived from gB-ORF8 help control infection

To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses.     

Apparent MHC-independent stimulation of CD8+ T cells in vivo during latent murine gammaherpesvirus infection.

Like EBV-infected humans with infectious mononucleosis, mice infected with the rodent gammaherpesvirus MHV-68 develop a profound increase in the number of CD8+ T cells in the circulation. In the mouse model, this lymphocytosis consists of highly activated CD8+ T cells strikingly biased toward V beta 4 TCR expression. Moreover, this expansion of V beta 4+CD8+ T cells does not depend on the MHC haplotype of the infected animal. Using a panel of lacZ-inducible T cell hybridomas, we have detected V beta 4-specific T cell stimulatory activity in the spleens of MHV-68-infected mice. We show that the appearance and quantity of this activity correlate with the establishment and magnitude of latent viral infection. Furthermore, on the basis of Ab blocking studies as well as experiments with MHC class II, beta2-microglobulin (beta2m) and TAP1 knockout mice, the V beta 4-specific T cell stimulatory activity does not appear to depend on conventional presentation by classical MHC class I or class II molecules. Taken together, the data indicate that during latent infection, MHV-68 may express a T cell ligand that differs fundamentally from both conventional peptide Ags and classical viral superantigens.     

Primary clearance of murine gammaherpesvirus 68 by PKCtheta-/- CD8 T cells is compromised in the absence of help from CD4 T cells

CD4 T cells are dispensable for acute control of murine gammaherpesvirus 68 (MHV-68) but are necessary for effective long-term control of the virus by CD8 T cells. In contrast, protein kinase C θ (PKCθ) is not essential for either acute or long-term viral control. However, we found that while either CD4 or CD8 T cells could mediate the clearance of MHV-68 from the lungs of PKCθ^+/+ mice, PKCθ^−/− mice depleted of either subset failed to clear the virus. These data suggest that there are two alternative pathways for MHV-68 clearance, one dependent on CD4 T cells and the other on PKCθ. Protection mediated by the latter appears to be short-lived. These observations may help to explain the differential requirement for PKCθ in various models of CD8 T-cell activation and differences in the costimulatory requirements for acute and long-term viral control.     

CD40 engagement on dendritic cells, but not on B or T cells, is required for long-term control of murine gammaherpesvirus 68

CD4 T cells are not essential for primary clearance of replicating murine gammaherpesvirus 68 (MHV-68) but are required for effective long-term control. The virus reactivates in the lungs of major histocompatibility complex class II-deficient (CII-/-) mice that lack functional CD4 T cells. CD40 ligand (CD40L) is upregulated on activated CD4 T cells, and it is thought that CD40-CD40L interactions are an important component of CD4 T-cell help. Our previous studies have shown that agonistic antibodies to CD40 can substitute for CD4 T-cell function in the long-term control of MHV-68. In the present study, we sought to identify the CD40-positive cell type mediating this effect. To address this question, we adoptively transferred MHV-68 peptide-pulsed CII(-/-) dendritic cells (DC) that had been treated with an agonistic antibody to CD40 into MHV-68-infected CII(-/-) recipients. Viral reactivation was significantly lower in mice injected with anti-CD40-treated DC than in those injected with control DC or in mice that did not receive any DC. However, in similar experiments with B cells, anti-CD40 treatment had no effect. We also investigated the requirement for CD40 expression on T cells by adoptive transfer of T cells from CD40(+/+) or CD40(-/-) mice into T-cell-deficient recipients that were subsequently infected with MHV-68. The results showed that CD40 expression on T cells is not necessary for preventing viral reactivation. Taken together, our data suggest that CD40 engagement on DC, but not on T or B cells, is essential for effective long-term control of MHV-68.     

Role of CXCR3 in the immune response to murine gammaherpesvirus 68

The chemokine IP-10 (CXCL10) and its cellular receptor CXCR3 are upregulated in the lung during murine gammaherpesvirus 68 (MHV-68) infection. In order to determine the role of the CXCR3 chemokine receptor in the immune response to MHV-68, CXCR3-/- mice were infected with the virus. CXCR3-/- mice showed delayed clearance of replicating MHV-68 from the lungs. This correlated with delayed T-cell recruitment to the lungs and reduced cytolytic activity prior to viral clearance. Splenomegaly and the numbers of latently infected cells per spleen were transiently increased. However, CXCR3-/- mice showed normal virus-specific antibody titers and effective long-term control of MHV-68 infection.     

CD4+ T cell priming accelerates the clearance of Sendai virus in mice, but has a negative effect on CD8+ T cell memory

Current vaccines designed to promote humoral immunity to respiratory virus infections also induce potent CD4+ T cell memory. However, little is known about the impact of primed CD4+ T cells on the immune response to heterologous viruses that are serologically distinct, but that share CD4+ T cell epitopes. In addition, the protective capacity of primed CD4+ T cells has not been fully evaluated. In the present study, we addressed these two issues using a murine Sendai virus model. Mice were primed with an HN421-436 peptide that represents the dominant CD4+ T cell epitope on the hemagglutinin-neuraminidase (HN) of Sendai virus. This vaccination strategy induced strong CD4+ T cell memory to the peptide, but did not induce Abs specific for the Sendai virus virion. Subsequent Sendai virus infection of primed mice resulted in 1) a substantially accelerated virus-specific CD4+ T cell response in the pneumonic lung; 2) enhanced primary antiviral Ab-forming cell response in the mediastinal lymph nodes; and 3) accelerated viral clearance. Interestingly, the virus-specific CD8+ T cell response in the lung and the development of long-term memory CD8+ T cells in the spleen were significantly reduced. Taken together, our data demonstrate that primed CD4+ T cells, in the absence of pre-existing Ab, can have a significant effect on the subsequent immune responses to a respiratory virus infection.     

Lytic cycle T cell epitopes are expressed in two distinct phases during MHV-68 infection.

Murine herpesvirus-68 (MHV-68) is a type 2 gamma herpesvirus that productively infects alveolar epithelial cells during the acute infection and establishes long-term latency in B cells and lung epithelial cells. In C57BL/6 mice, T cells specific for lytic cycle MHV-68 epitope p56/Db dominate the acute phase of the infection, whereas T cells specific for another lytic cycle epitope, p79/Kb, dominate later phases of infection. To further understand this response, we analyzed the kinetics of Ag presentation in vivo using a panel of lacZ-inducible T cell hybridomas specific for several lytic cycle epitopes, including p56/Db and p79/Kb. Two distinct peaks of Ag presentation were observed. The first peak was at day 6 in the mediastinal lymph nodes and correlated with the initial pulmonary lytic infection. The second peak was at day 18 in both the mediastinal lymph nodes and spleen and correlated with the peak of latent infection. Interestingly, the p56 epitope was detected only in the mediastinal lymph nodes at day 6 after infection whereas the p79 epitope was predominantly presented in the spleen at day 18, suggesting that differential epitope presentation drives the switch in T cell responses to this virus. Phenotypic analysis of APCs at day 18 postinfection revealed that dendritic cells, macrophages, and B cells all presented lytic cycle epitopes. Taken together, the data suggest that there is a resurgence of lytic cycle Ags in the spleen, which explains the kinetics and specificity of the T cell response to MHV-68.